The Human Immunodeficiency Virus
The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). HIV causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in their depletion. This, in turn, leads to opportunistic infections, neoplastic growth and death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, 1988, Science 240:1427-1439). There are at least two distinct subtypes of HIV: HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). Genetic heterogeneity exists within each of these HIV subtypes.
CD4.sup.+ T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon viral protein gp120 binding to the cellular CD4 receptor molecule (McDougal et al., 1986,Science 231:382-385; Maddon et al., 1986, Cell 47:333-348).
HIV Treatment
HIV infection is pandemic and HIV-associated diseases have become a world-wide health problem. Despite considerable efforts in the design of anti-HIV modalities, there is, thus far, no successful prophylactic or therapeutic regimen against AIDS. However, several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al., 1991, FASEB J. 5:2369-2381). For example, virally-encoded reverse transcriptase has been a major focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2',3'-dideoxynucleotide analogs such as AZT, ddI, ddC, and ddT have been shown to be active against HIV (Mitsuya et al., 1990, Science 249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al., 1989, Science 243:1731-1734). In addition, these drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
Another stage of the HIV life cycle that has been targeted is viral entry into the cells, the earliest stage of HIV infection. This approach has primarily utilized recombinant soluble CD4 protein to inhibit infection of CD4.sup.+ T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). To date, clinical trials of recombinant, soluble CD4 have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med. 112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
Additionally, the later stages of HIV replication which involve crucial virus-specific secondary processing of certain viral proteins and enzymes have been targeted for anti-HIV drug development. Late stage processing is dependent on the activity of a virally-encoded protease, and drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al., 1993, Persp. Drug. Discov. Design 1:69-83). With this class of drugs, the emergence of drug resistant HIV mutants is also a problem; resistance to one inhibitor often confers cross resistance to other protease inhibitors (Condra et al., 1995, Nature 374:569-571). These drugs often exhibit toxic side effects such as nausea, altered taste, circumoral parethesias and nephrolithiasis.
Antiviral therapy of HIV using different combinations of nucleoside analogs and protease inhibitors have recently been shown to be more effective than the use of a single drug alone (Torres et al., 1997, Infec. Med. 14:142-160). However, despite the ability to achieve significant decreases in viral burden, there is no evidence to date that combinations of available drugs will afford a curative treatment for AIDS.
Other potential approaches for developing treatment for AIDS include the delivery of exogenous genes into infected cells. One such gene therapy approach involves the use of genetically-engineered viral vectors to introduce toxic gene products to kill HIV-infected cells. Another form of gene therapy is designed to protect virally-infected cells from cytolysis by specifically disrupting viral replication. Stable expression of RNA-based (decoys, antisense and ribozymes) or protein-based (transdominant mutants) HIV-1 antiviral agents can inhibit certain stages of the viral life cycle. A number of anti-HIV suppressors have been reported, such as decoy RNA of TAR or RRE (Sullenger et al., 1990, Cell 63:601-608; Sullenger et al., 1991, J. Virol. 65:6811-6816; Lisziewicz et al., 1993, New Biol. 3:82-89; Lee et al., 1994, J. Virol. 68:8254-8264), ribozymes (Sarver et al., 1990, Science 247:1222-1225; Wecrasinghe et al., 1991, J. Virol. 65:5531-5534; Dropulic et al., 1992, J. Virol. 66:1432-1441; Ojwang et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10802-10806; Yu et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6340-6344; Yu et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:699-703; Yamada et al., 1994, Gene Therapy 1:38-45), antisense RNA complementary to the mRNA of gag, tat, rev, env (Sezakiel et al., 1991, J. Virol. 65:468-472; Chatterjee et al., 1992, Science 258:1485-1488; Rhodes et al., 1990, J. Gen. Virol. 71:1965; Rhodes et al.,1991, AIDS 5:145-151; Sezakiel et al., 1992, J. Virol. 66:5576-5581; Joshi et al., 1991, J. Virol. 65:5524-5530) and transdominant mutants including Rev (Bevec et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:9870-9874), Tat (Pearson et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:5079-5083; Modesti et al., 1991, New Biol. 3:759-768), Gag (Trono et al., 1989, Cell 59:113-120), Env (Bushschacher et al., 1995, J. Virol. 69:1344-1348) and protease (Junker et al., 1996, J. Virol. 70:7765-7772).
Antisense polynucleotides have been designed to complex with and sequester the HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al., WO 94/10302; Kretschmer et al., EP 594,881; and Chatterjee et al., 1992, Science 258:1485). Furthermore, an enzymatically active RNA, termed ribozyme, has been used to cleave viral transcripts. The use of a ribozyme to generate resistance to HIV-1 in a hematopoietic cell line has been reported (Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89:10802-06; Yamada et al., 1994, Gene Therapy 1:38-45; Ho et al., WO 94/26877; and Cech and Sullenger, WO 95/13379). In preclinical studies, RevM10, a transdominant Rev protein, has been transfected ex vivo into CD4.sup.+ cells of HIV-infected individuals and shown to confer survival advantage over cells transfected with vector only (Woffendin et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2889-2894).
Cellular Genes Necessary For HIV Replication
Evolution of an intracellular pathogen has resulted in the development of interactions of its genes and gene products with multiple cellular components. For instance, the interactions of a virus with a host cell involves binding of the virus to a specific cellular receptor(s), translocation through the cellular membrane, uncoating, replication of the viral genome, transcription of the viral genes, etc. Each of these events occurs in a cell and involves interactions with a cellular component. Thus, the life cycle of a virus can be completed only if the cell is permissive. Availability of amino acids and nucleotides for replication of the viral genome and protein synthesis, energy status of the cell, the presence of cellular transcription factors and enzymes all contribute to the propagation of the virus in the cell. Consequently, the cellular components, in part, determine host cell susceptibility to infection, and may be used as potential targets for the development of new therapeutic interventions. In the case of HIV, one cellular component which has been used towards this end is the cell surface molecule for HIV attachment, CD4.
Recently, it was reported that HIV entry into a susceptible cell requires the expression of a second receptor, the chemokine receptor (CCR5 or CXCR4), in addition to CD4 (Moore, 1997, Science 276:51-52). A chemokine receptor normally binds RANTES, MIP-1.alpha. and MIP-1.beta. as its natural ligand. In the case of HIV infection, it has been proposed that CD4 first binds to gp120 of HIV on the cell surface followed by the binding of the complex to a chemokine receptor to result in viral entry into the cells (Cohen, 997, Science 275:1261). Therefore, chemokine receptors may present an additional cellular target for the design of HIV therapeutic agents. Inhibitors of HIV/chemokine receptor interactions are being tested as anti-HIV agents. However, there remains a need for the discovery of additional cellular targets for the design of anti-HIV therapeutics, particularly intracellular targets for disrupting viral replication after viral entry into a cell.